ChIP-on-Chip Analysis of In Vivo Mutant p53 Binding To Selected Gene Promoters Stefania Dell’Orso, 1, * Giulia Fontemaggi, 1,2,3, * Perry Stambolsky, 4 Frauke Goeman, 1,2 Christine Voellenkle, 5 Massimo Levrero, 6 Sabrina Strano, 7 Varda Rotter, 4 Moshe Oren, 4 and Giovanni Blandino 1,2 Abstract Growing evidence shows that mutant p53 proteins, which are present in many human tumors, gain oncogenic activities that can actively contribute to tumorigenesis. Mutant p53 proteins have been extensively shown to affect the expression of several genes involved in various aspects of cancer biology. We show here the ChIP-on-chip analysis of mutant p53 binding to a set of 154 promoters, composed of both validated and putative mutant p53 target genes. By using the chromatin obtained from mutant p53R175H-immunoprecipitation in proliferating SKBr3 breast cancer cells, we found that mutant p53 binds to 40 of the 154 promoters analyzed. siRNA-mediated mutant p53 knock-down modulates the transcript abundance of some of these target genes. Two-thirds of the mutant p53- bound promoters were also engaged by either p300 or PCAF acetyl-transferases, strongly indicating the presence of transcriptionally active complexes. We also found that NF-kB binding sites are overrepresented among the mutant p53-bound promoters; a ChIP-on-chip analysis confirmed that NF-kB p65 binds to 27 of the mutant p53-bound promoters, indicating that mutant p53 could influence the transcriptional output of these NF-kB target genes. Introduction C ancer-associated mutant p53 proteins result mainly from missense mutations that frequently occur in the TP53 gene region encoding for the DNA-binding domain (DBD). The resulting full-length proteins are abundantly present in tumor cells. In vitro and in vivo evidence implies that mutant p53 proteins do not only represent the mere loss of antitumoral wild-type p53 (wtp53) activities but also ac- quire a gain of oncogenic functions through which they ac- tively participate in the establishment, dissemination, and resistance to conventional anticancer treatments of human tumors. Such activities are commonly described as mutant p53 gain-of-function (GOF) (reviewed in Brosh and Rotter, 2009; Oren and Rotter, 2010). TP53 mutations are more fre- quent in advanced stage cancer or in cancer subtypes with aggressive behavior (Langerod et al., 2007; Wang et al., 2004a, 2004b) and have been consistently associated with poor prognosis in cancers such as breast, colorectal, head and neck, and leukemia (Petitjean et al., 2007). The molecular mechanisms underlying gain of function of mutant p53 proteins still remain to be fully elucidated. Two- thirds of missense mutations in the DNA-binding domain, including all hotspot mutations, abrogate the ability of p53 to bind and transactivate canonical wild-type p53 binding sites; despite that, mutant p53 proteins have been shown to mod- ulate gene expression and were recently found recruited to the regulatory regions of genes with important roles in cancer biology. These findings raise the possibility that mutant p53 functions as an oncogenic transcription factor capable to ab- errantly modulate gene expression (reviewed in Strano et al., 2007). A unifying mechanism for the selectivity of mutant p53 toward certain genes is still missing, owing to the lack of a consensus DNA sequence among genes regulated by mutant p53 and the variability in the identity of the genes affected by different p53 mutants. We and others have previously re- ported that mutant p53 reaches target gene promoters through the interaction with sequence-specific transcription factors, such as NF-Y, E2F1, NF-kB, and Vitamin D receptor (Di Agostino et al., 2006; Fontemaggi et al., 2009, 2010; 1 Translational Oncogenomics Unit, Regina Elena Cancer Institute, Rome, Italy. 2 Rome Oncogenomic Center (ROC), Regina Elena Cancer Institute, Rome, Italy. 3 General Pathology Section, Department of Clinical and Experimental Medicine, Perugia University, Perugia, Italy. 4 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 5 Research Laboratories-Molecular Cardiology, Policlinico San Donato, S. Donato M.se (MI) Italy. 6 Department of Internal Medicine, Sapienza Universita ` , Rome, Italy. 7 Molecular Chemoprevention Group, Scientific Direction, Regina Elena Cancer Institute, Rome, Italy. *These authors contributed equally to this article. OMICS A Journal of Integrative Biology Volume 15, Number 5, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2010.0084 305